Nitrogen (N) is the most abundant inorganic nutrient taken up from the soil by plants for growth and development. Maize roots absorb most of the N from the soil in the form of nitrate, the majority of which is transported to the leaf for reduction and assimilation. Nitrate is reduced to nitrite by nitrate reductase (NR) in the cytosol and then nitrite is transported into chloroplast where it is reduced by nitrite reductase (NiR) to ammonium. Ammonium is assimilated into glutamine by the glutamine synthase-glutamate synthase system (Crawford and Glass, (1998) Trends in Plant Science 3:389-395). Also, it has long been known that significant amounts of N are lost from the plant aerial parts by volatilization (Glyan'ko, et al., (1980) Agrokhimiya 8:19-26; Hooker, et al., (1980) Agronomy Journal 72(5):789-792; Silva, et al., (1981) Crop Science 21(6):913-916; Stutte, et al., (1981) Crop Science 21(4):596-600; Foster, et al., (1986) Annals of Botany 57(3):305-307; Parton, et al., (1988) Agronomy Journal 80(3):419-425; Kamiji, et al., (1989) Japanese Journal of Crop Science 58(1):140-142; Morgan, et al., (1989) Crop Science 29(3):726-731; O'Deen, (1989) Agronomy Journal 81(6):980-985; Guindo, et al., (1994) Arkansas Farm Research 43(1):12-13; Heckathorn, et al., (1995) Oecologia 101(3):361-365; Cabezas, et al., (1997) Revista Brasileira de Ciencia do Solo 21(3):481-487). Experimental evidence supports the loss of N through ammonium and not through N oxides (Hooker, et al., 1980). Treatment with chemicals that inhibit glutamine or glutamate synthase activities led to increased loss of ammonium through volatilization (Foster, et al., 1986). Loss of N is not only limited to C-3 species as C-4 plants have also been reported to lose N through volatilization (Heckathorn, et al., 1995).
Several independent lines of evidence indicate that glutamine synthetase (GS) is involved in yield formation and its expression levels affect nitrogen use efficiency (NUE) in maize. GS carries out two main functions in plant cells: (1) assimilate ammonium resulting from nitrate reduction into organic form during the biosynthetic phase and (2) assimilate ammonium generated by photorespiration, deaminases and glutamate dehydrogenase, for example, during seed germination and leaf senescence when proteins are remobilized as N source or used as source of energy. The cytosolic GS is referred to as GS1 and the plastidial form as GS2. In a recent report (Martin, et al., (2006) The Plant Cell 18(11):3252-74), a reverse genetics strategy was used to show that GS indeed is a limiting factor for grain number and grain weight, both components of grain yield in maize. Earlier QTL mapping experiments also implicated GS isozymes in the determination of yield and NUE (Gallais and Hirel, (2004) J Exp Bot. 55(396):295-306). In other experiments, two GS genes located on chromosome 1, including one expressed in the root, show significant (p=10−4) association with biomass at 1 and 5 mM applied N (data not shown). During leaf senescence, remobilization of N takes place from source (leaf) to sink (developing grain) tissues. Proteins are broken down into amino-acids, which are then transported through phloem to the sink tissue. Grain protein accounts for ˜60-70% of the total plant N at maturity in maize, which means 30-40% N still remains in the stover. The current invention involves efforts to over-express the cytosolic isoforms of GS under the control of different promoters in maize to improve NUE and thus grain yield.